Activated carrier molecules and their relationship to enzymes

Activated carrier molecules and their relationship to enzymes

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I am reading Molecular Biology of the Cell, and one thing I don't quite get is the difference between an enzyme and an activated carrier molecule. I understand that enzymes lower the activation energy for a reaction, and in that sense speed up the rate at which a reaction occurs. I also understand that activated carrier molecules act as a source of energy that can drive unfavorable reactions (like anabolic reactions) forward. But I still don't quite grasp the exact difference. I was wondering if someone could shed some light on this.

I will focus on the meaning of 'activated carrier molecule' as descriptions of enzymes abound. In order to do that I need to introduce two other ideas first. I apologize to the purists for tailoring this to the level of the question.

1. Gibbs Free Energy in Biochemical Considerations

There are different ways of considering energy, and in chemistry one may be used to considering bond energy in relation to a particular molecule. However in biochemistry (which is what this question is concerned with) the focus is on the energetics of overall processes. For example “Are the reactions involved in the synthesis of glucose from pyruvate energetically favourable?" or “How can the synthesis of glucose from pyruvate occur if the overall reaction is not energetically favourable?” Likewise for individual reactions, such as the formation of peptide bonds between amino acids.

For considerations of this sort the thermodynamical concept (Gibbs) Free Energy (G) is most useful. This is because the change in free energy (ΔG) during a reaction (or other change of state) indicates whether a reaction can occur spontaneously. To quote from a useful section in Berg et al.

A reaction can occur spontaneously only if ΔG is negative.

So in the reaction sequence just mentioned, the overall synthesis of glucose from pyruvate (gluconeogenesis) has a positive ΔG and is energetically unfavourable, whereas that of the reverse process (glycolysis) is energetically favourable. And likewise peptide bond formation, not surprisingly, has a positive ΔG and is not energetically favourable.

2. How are Energetically Unfavourable Reactions Accomplished in Living Cells?

The question posed in the heading arises from the fact that gluconeogenesis and peptide bond formation occur in living cells, even though taken alone the core reactions that represent the processes are energetically unfavourable. The answer is given in the title of [section 14.1.1. of Berg et al.]

A Thermodynamically Unfavourable Reaction can be driven by a Favourable Reaction

As already stated, it is the ΔG of the overall process that is important, so if a reaction with a positive ΔG is 'coupled' to one of negative ΔG of greater magnitude, the overall reaction can proceed. For example:

A → B ΔG = 20 kJ/mol (unfavourable) C → D ΔG = -30 kJ/mol (favourable) A + C → B + D ΔG = -10 kJ/mol (favourable)

If reaction C → D proceeds alone, the free energy is liberated as biochemically useless heat. If it is coupled to reaction A → B it drives the latter with the balance being lost as heat.

3. The Role of Activated Carrier Molecules in Energy Coupling

Clearly, any coupled reaction can cause water to run uphill, as it were. But in order to continue one needs to regenerate the starting molecules for the favourable reaction. It is more efficient to specialize this regeneration process by using a limited range of specialist reactions with a negative free energy change. One description of the substrates (reacting molecules) of such specialized favourable reactions is “activated carrier molecules”. Alberts et al. define them as:

Small diffusible molecules in cells that store easily-exchangeable energy in the form of one or more energy-rich covalent bonds. Examples are ATP and NADPH.

Berg et al. also use the term, but describe it in terms of the following examples:

  • ATP as an activated carrier of phosphoryl groups, because phosphoryl transfer from ATP is an exergonic process (i.e. has a negative ΔG)
  • Nicotinamide adenine dinucleotide (NAD+) is a major electron carrier in the oxidation of fuel molecules. (Although in this case it is the half reaction involving the oxidation of the reduced form, NADH - or NADPH as mentioned by Alberts et al. - that has a negative ΔG).
  • Coenzyme A… is a carrier of acyl groups (but it is the transfer of the acyl group of acyl-CoA molecules - e.g. acetyl CoA - with the generation of CoA that has a negative ΔG).

Examples of the participation of such molecules in metabolism can be found in the volume referenced, however I would emphasize that it is the specific reactions of these molecules involving the groups (or electrons) that they carry that is important.

And what about Enzymes?

As the poster stated “enzymes lower the activation energy for a reaction”. They are (generally) large proteins catalysts that have no effect on the ΔG, and they are unchanged at the end of the reaction.

Of course they are important for biochemical reactions, they participate in them, and provide binding sites to bring the substrates and activated carriers in juxtaposition so that they can react. But they couldn't be more different from the small activated carriers.

4.2: Control of Enzymatic Activity

  • Contributed by Kevin Ahern, Indira Rajagopal, & Taralyn Tan
  • Professor (Biochemistry and Biophysics) at Oregon State University

A printable version of this section is here: BiochemFFA_4_2.pdf. The entire textbook is available for free from the authors at

Methods of Intracellular Signaling

Signaling pathway induction activates a sequence of enzymatic modifications that are recognized in turn by the next component downstream.

Learning Objectives

Explain how the binding of a ligand initiates signal transduction throughout a cell

Key Takeaways

Key Points

  • Phosphorylation, the addition of a phosphate group to a molecule such as a protein, is one of the most common chemical modifications that occurs in signaling pathways.
  • The activation of second messengers, small molecules that propagate a signal, is a common event after the induction of a signaling pathway.
  • Calcium ion, cyclic AMP, and inositol phospholipids are examples of widely-used second messengers.

Key Terms

  • second messenger: any substance used to transmit a signal within a cell, especially one which triggers a cascade of events by activating cellular components
  • phosphorylation: the addition of a phosphate group to a compound often catalyzed by enzymes

The induction of a signaling pathway depends on the modification of a cellular component by an enzyme. There are numerous enzymatic modifications that can occur which are recognized in turn by the next component downstream.

One of the most common chemical modifications that occurs in signaling pathways is the addition of a phosphate group (PO4 –3 ) to a molecule such as a protein in a process called phosphorylation. The phosphate can be added to a nucleotide such as GMP to form GDP or GTP. Phosphates are also often added to serine, threonine, and tyrosine residues of proteins where they replace the hydroxyl group of the amino acid. The transfer of the phosphate is catalyzed by an enzyme called a kinase. Various kinases are named for the substrate they phosphorylate. Phosphorylation of serine and threonine residues often activates enzymes. Phosphorylation of tyrosine residues can either affect the activity of an enzyme or create a binding site that interacts with downstream components in the signaling cascade. Phosphorylation may activate or inactivate enzymes the reversal of phosphorylation, dephosphorylation by a phosphatase, will reverse the effect.

Example of phosphorylation: In protein phosphorylation, a phosphate group (PO4-3 ) is added to residues of the amino acids serine, threonine, and tyrosine.

The activation of second messengers is also a common event after the induction of a signaling pathway. They are small molecules that propagate a signal after it has been initiated by the binding of the signaling molecule to the receptor. These molecules help to spread a signal through the cytoplasm by altering the behavior of certain cellular proteins.

Calcium ion is a widely-used second messenger. The free concentration of calcium ions (Ca 2+ ) within a cell is very low because ion pumps in the plasma membrane continuously use adenosine-5′-triphosphate ( ATP ) to remove it. For signaling purposes, Ca 2+ is stored in cytoplasmic vesicles, such as the endoplasmic reticulum, or accessed from outside the cell. When signaling occurs, ligand-gated calcium ion channels allow the higher levels of Ca 2+ that are present outside the cell (or in intracellular storage compartments) to flow into the cytoplasm, which raises the concentration of cytoplasmic Ca 2+ . The response to the increase in Ca 2+ varies, depending on the cell type involved. For example, in the β-cells of the pancreas, Ca 2+ signaling leads to the release of insulin, whereas in muscle cells, an increase in Ca 2+ leads to muscle contractions.

Another second messenger utilized in many different cell types is cyclic AMP (cAMP). Cyclic AMP is synthesized by the enzyme adenylyl cyclase from ATP. The main role of cAMP in cells is to bind to and activate an enzyme called cAMP-dependent kinase (A-kinase). A-kinase regulates many vital metabolic pathways. It phosphorylates serine and threonine residues of its target proteins, activating them in the process. A-kinase is found in many different types of cells the target proteins in each kind of cell are different. Differences give rise to the variation of the responses to cAMP in different cells.

Example of cAMP as a second messenger: This diagram shows the mechanism for the formation of cyclic AMP (cAMP). cAMP serves as a second messenger to activate or inactivate proteins within the cell. Termination of the signal occurs when an enzyme called phosphodiesterase converts cAMP into AMP.

Present in small concentrations in the plasma membrane, inositol phospholipids are lipids that can also be converted into second messengers. Because these molecules are membrane components, they are located near membrane-bound receptors and can easily interact with them. Phosphatidylinositol (PI) is the main phospholipid that plays a role in cellular signaling. Enzymes known as kinases phosphorylate PI to form PI-phosphate (PIP) and PI-bisphosphate (PIP2).

Biosynthesis and structure–activity relationships of the lipid a family of glycolipids

The glycolipid Lipid A is the conserved, amphipathic moiety of LPS.

Lipid A binds to certain Toll like receptors and caspases with high affinity.

Bacteria alter their Lipid A structures to modulate immune activities.

Non-toxic Lipid A congeners are effective immunomodulatory agents.

Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram-negative bacteria, is a potent elicitor of innate immune responses in mammals. A typical LPS molecule is composed of three different structural domains: a polysaccharide called the O-antigen, a core oligosaccharide, and Lipid A. Lipid A is the amphipathic glycolipid moiety of LPS. It stimulates the immune system by tightly binding to Toll-like receptor 4. More recently, Lipid A has also been shown to activate intracellular caspase-4 and caspase-5. An impressive diversity is observed in Lipid A structures from different Gram-negative bacteria, and it is well established that subtle changes in chemical structure can result in dramatically different immune activities. For example, Lipid A from Escherichia coli is highly toxic to humans, whereas a biosynthetic precursor called Lipid IVA blocks this toxic activity, and monophosphoryl Lipid A from Salmonella minnesota is a vaccine adjuvant. Thus, an understanding of structure–activity relationships in this glycolipid family could be used to design useful immunomodulatory agents. Here we review the biosynthesis, modification, and structure–activity relationships of Lipid A.

Enzymes: How they work and what they do

Enzymes help speed up chemical reactions in the human body. They bind to molecules and alter them in specific ways. They are essential for respiration, digesting food, muscle and nerve function, among thousands of other roles.

In this article, we will explain what an enzyme is, how it works, and give some common examples of enzymes in the human body.

Share on Pinterest The enzyme amylase (pictured), breaks down starch into sugars.

Enzymes are built of proteins folded into complicated shapes they are present throughout the body.

The chemical reactions that keep us alive – our metabolism – rely on the work that enzymes carry out.

Enzymes speed up (catalyze) chemical reactions in some cases, enzymes can make a chemical reaction millions of times faster than it would have been without it.

A substrate binds to the active site of an enzyme and is converted into products. Once the products leave the active site, the enzyme is ready to attach to a new substrate and repeat the process.

The digestive system – enzymes help the body break down larger complex molecules into smaller molecules, such as glucose, so that the body can use them as fuel.

DNA replication – each cell in your body contains DNA. Each time a cell divides, that DNA needs to be copied. Enzymes help in this process by unwinding the DNA coils and copying the information.

Liver enzymes – the liver breaks down toxins in the body. To do this, it uses a range of enzymes.

The “lock and key” model was first proposed in 1894. In this model, an enzyme’s active site is a specific shape, and only the substrate will fit into it, like a lock and key.

This model has now been updated and is called the induced-fit model.

In this model, the active site changes shape as it interacts with the substrate. Once the substrate is fully locked in and in the exact position, the catalysis can begin.

Enzymes can only work in certain conditions. Most enzymes in the human body work best at around 37°C – body temperature. At lower temperatures, they will still work but much more slowly.

Similarly, enzymes can only function in a certain pH range (acidic/alkaline). Their preference depends on where they are found in the body. For instance, enzymes in the intestines work best at 7.5 pH, whereas enzymes in the stomach work best at pH 2 because the stomach is much more acidic.

If the temperature is too high or if the environment is too acidic or alkaline, the enzyme changes shape this alters the shape of the active site so that substrates cannot bind to it – the enzyme has become denatured.

Some enzymes cannot function unless they have a specific non-protein molecule attached to them. These are called cofactors. For instance, carbonic anhydrase, an enzyme that helps maintain the pH of the body, cannot function unless it is attached to a zinc ion.

To ensure that the body’s systems work correctly, sometimes enzymes need to be slowed down. For instance, if an enzyme is making too much of a product, there needs to be a way to reduce or stop production.

Enzymes’ activity can be inhibited in a number of ways:

Competitive inhibitors – a molecule blocks the active site so that the substrate has to compete with the inhibitor to attach to the enzyme.

Non-competitive inhibitors – a molecule binds to an enzyme somewhere other than the active site and reduces how effectively it works.

Uncompetitive inhibitors – the inhibitor binds to the enzyme and substrate after they have bound to each other. The products leave the active site less easily, and the reaction is slowed down.

Irreversible inhibitors – an irreversible inhibitor binds to an enzyme and permanently inactivates it.

There are thousands of enzymes in the human body, here are just a few examples:

  • Lipases – a group of enzymes that help digest fats in the gut.
  • Amylase – helps change starches into sugars. Amylase is found in saliva.
  • Maltase – also found in saliva breaks the sugar maltose into glucose. Maltose is found in foods such as potatoes, pasta, and beer.
  • Trypsin – found in the small intestine, breaks proteins down into amino acids.
  • Lactase – also found in the small intestine, breaks lactose, the sugar in milk, into glucose and galactose.
  • Acetylcholinesterase – breaks down the neurotransmitter acetylcholine in nerves and muscles.
  • Helicase – unravels DNA.
  • DNA polymerase – synthesize DNA from deoxyribonucleotides.

Enzymes play a huge part in the day-to-day running of the human body. By binding to and altering compounds, they are vital for the proper functioning of the digestive system, the nervous system, muscles, and much, much more.

Evolution of First Cell | Biology

Early living cells were RNA life forms, self- replicating RNA covered by lipoprotein vesicles were the pre-prokaryotes, with time the proteins replaced the catalytic function of RNA, and DNA replaced the coding function of RNA, the progenitors of modern prokaryotes with DNA-RNA-protein functioning types evolved.

Evolution of first primitive cell from RNA world represents a huge gap. Primitive bacterial cell represents an immensely complicated struc­ture with at least 1000 genes in comparison with our ideas about RNA world.

Following problems need to be solved to fill this gap:

1. Dominating role of protein as enzymes over ribozymes.

2. Differentiation of different types of RNA.

3. The shift from RNA to DNA as carrier of genetic information.

5. Formation of chromosome.

6. Increasing genetic information.

7. Phenotypic expression of a genotype.

9. Evolution of metabolic process.

Introduction of pro­teins as enzymes resulted in more specific cata­lysis. Enzymatic capability of the RNA strands could be improved if individual amino acids were attached as in tRNA, i.e., amino acids acted as co-enzymes for the ribozymes. The next step is the specialization of RNA so that ‘+’ strand had the role as mRNA and ‘-‘ strand functioned as tRNA and attached to the ‘+’ strand with an anti-codon triplet.

Finally, the amino acids could be coupled together as a polypeptide strand that would further improve catalytic activity. This idea is supported by the fact that tRNAs from different organisms with similar function are more closely related and thus tRNA can be traced back to the origin of the genetic code and to a RNA world.

Differentiation of Different Types of RNA:

Functional specialization of different RNAs was adaptive in increasing the efficiency within proto-cells. Some kinds of RNA (tRNA) specialized in collecting amino acids and others (rRNA) in cou­pling them together are the basis of the code in a third kind of RNA (mRNA).

Emergence of complex organisms requires the transition from RNA to DNA as genetic material. Double stranded DNA is much more stable than RNA and allows enzy­matic proof-reading and correction in connection with replication and thus reduces the rate of mutation (Fig. 2.10).

The genetic information in RNA organisms corresponds to a maximum of 10 thousand base pairs in comparison to 10 million base pairs in bacterial chromosome.

Replacement of ribose by deoxyribose in the carbohydrate backbone of RNA and replacement of uracil base by thymine resulted in DNA. Deoxyribose is formed in cells through an enzymatically con­trolled reduction of ribose. Enzymatic synthesis of DNA from RNA by reverse transcriptase in RNA virus is well known today.

Origin of Code in First Cell:

Genetic code based on four bases expressed in triplets with redundancy for twenty amino acids is almost universal. Though there is no chemical relationship between the mRNA codon or anticodon of tRNA and the chemical structure of amino acid, but the speci­ficity of given tRNA to a particular amino acid has developed. All these features minimizes the risk of replication errors and rate of point mutations.

Formation of Chromosome:

Free floating RNA molecules once enclosed in a membrane would become adaptive to have genes linked together in a single chromosome. Different kinds of free-floating RNA molecules replicated inside their proto-cells undergo unequal distribution in daughter cells after division of proto-cell with reduced fitness.

This might be overcome by con­necting the RNA molecules into a single strand combined with simultaneous replication which results in equal distribution of genome between daughter cells.

Increasing Genetic Information:

Genome size gets increased with increasing complexity from a couple of genes in virus to 1000 in bacte­ria, 5000 in fruit-fly, and 30 000 in human or higher plants, but not associated with a drastic increase in the number of translatable genes.

The important mechanism of increasing genetic information is gene doubling followed by muta­tion and selection leading to production of new enzyme and biomolecules. Natural horizontal gene transfer as found in bacteria (transforma­tion, conjugation, transduction) could lead to increase in genetic information.

Phenotypic Expression of a Genotype:

Though genes are often correlated to certain phenotypic traits but genes only specify proteins/ enzymes. Variations of a gene (alleles) can have effects on the phenotype through variations in the specified protein. Actually the production of a given phenotype is the result of network of interactions between genes and enzymes and between different enzymes which is far too com­plex to be un-ravelled.

Origin of Cell Membrane in First Cell:

Spontaneous for­mation of molecular double layer on the water surfaces by lipids served as a model for the ori­gin of double layer phospholipid cell membrane. This is due to hydrophobic (mutually attracted) and hydrophilic (attracted to water) end of linear-molecules (Fig. 2.11).

If the lipid films form spheres, the hydrophobic ends are hidden inside the film attaining lowest energy state. Phospho­lipids are easily formed in the presence of lipids, glycerol and phosphate and such spheres can be made experimentally through shaking and sonication.

Constant addition of mass to the contents of the spheres and to the membrane results in budding and division of cells. Residence of most vital functions (energy metabolism, transport channels) of the cell in the cell membrane is based on a variety of embedded protein (Fig. 2.12).

Evolution of Metabolism in First Cell:

The fundamental types of energy metabolism are photo-trophy, respiration, fermentation, metanogenesis all of which are represented among the bacteria. Dissimilatory energy metabolism (catabolic) refers to the mechanism to generate ATP with high energy rich phosphate bonds (Fig. 2.13).

Assimilatory metabolism (anabolic) refers to metabolic processes that serve to build the com­ponents of the cell from chemical compounds of environment through phototrophic (photosynthe­sis), chemotrophic (chemosynthesis) or hetero­trophic modes. The process of energy meta­bolism are based on coupled redox processes of the type AH2 -1- B BH2 + A.

The important hydrogen carrier found in cell is NADVNADFH or its phosphorylated version NADP/NADPH (Fig. 2.14).

Fermentation represented the most primitive form of energy metabolism whose biochem­istry is simple and does not require an external oxidant (electron acceptor) and independent of O2. Well known fermentation processes include lactic acid fermentation, ethanol fermentation, butyric acid fermentation. Respiratory carbohy­drate metabolism is initiated by an anaerobic fer­mentation.

First membrane bound electron trans­port mechanism was based on simple functional molecules but without the protein component. The protein component developed later which improved efficiency and specificity.

Such naked molecules like quinine, metal containing por­phyrins, inorganic FeS common in anoxic prebi­otic earth, could have incorporated into primitive cell membrane that can be photo-activated and responsible for a primitive electron transport system or a kind of photochemical energy trans­duction (Fig. 2.15).

Photosensitive porphyrin has become protochlorophyll and cytochrome. Respiring organisms are derived from phototrophic one through secondary loss of chloro­phylls and dependent on external chemical reductants. The photosynthetic purple non- sulphur bacteria has electron transport system almost identical to that of mitochondria (Fig. 2.16).

Mechanism to explain the origin of compli­cated biochemical processes involving many steps and cycles is the fact that these pathways are mostly reversible, catalysing the process in either direction.

Assimilatory reduction of CO2 with the help of NADFH2 and energy (ATP) may run in a opposite way and become dissimilatory and oxidative pathway, degrading and oxidizing organic matter into CO2 and release ATP through respiratory glycolytic pathway and citric acid cycle (Fig. 2.17).

CO2 assimilated through Calvin cycle into organic matter undergoes oxidation through glycolytic pathway which is actually a reverse process of Calvin cycle. The origin of citric acid cycle can be traced by the fact that green sulphur bacteria assimilate CO2 through a reverse citric acid cycle which is reductive and requires ATP (Fig. 2.18).

Prokaryotic Cell:

From the above discussion it is crystal clear that the chemical evolution on prebiotic earth gave rise to organic molecules which included protein, nucleic acid, etc. establishment of tem­plate system evolved enzyme systems and a sur­rounding lipid membrane an energy transfer mechanism involving ATP has evolved.

This may have been the beginning of a stable structural and functional organisation having resemblance of a biological cell. These cells are called prokaryotic because of the absence of membrane bound nucleus and organelles.

Primitive prokaryotic cells were essentially anaerobic cells (anaerobic bacteria) because the early earth was devoid of oxygen. Depletion of organic compounds in the primaeval soup resulted in the appearance of photosynthetic cells (blue green algae) which can fix CO2 and probably nitrogen also.

Photosynthetic cells were responsible for production of oxygen in atmosphere which resulted in the ori­gin of aerobic cells (aerobic bacteria) with metabolic pathways for aerobic respiration.

Enzyme Production and Purification: Extraction & Separation Methods | Industrial Microbiology

In this article we will discuss about the production and purification of enzymes. Learn about the e xtraction and separation methods for isolation and purification of enzymes. The extraction methods are: 1. Extraction of Solid Substrate Cultures 2. Extraction of Cells and the separation methods are: 1. Solids Separation Techniques 2. Membrane Separation Techniques 3. Gel Filtration 4. Adsorption Techniques 5. Precipitation Techniques. And also glance over the below given article to get an idea about s torability of enzymes and e nzyme immobilization .

In enzyme production there is a very unfavorable ratio between input of raw material and output of product. This requires the installation of con­centration procedures. For economic reasons of enzyme application a con­centration up to 10-fold is usually satisfactory for industrial enzyme prepa­rations.

For example- enzyme products employed in detergents contain about 5-10% protease while amylase preparations for use in flour treat­ment contain only about 0.1% pure a-amylase. However, in applications where high purity enzymes are required, e.g., in enzymic analysis, 1000- fold purification is quite common.

In some applications, such as baking and dextrose manufacture, the presence of contaminating enzymes must be very low or rigidly controlled. Moreover, the raw enzyme solutions obtained from microbial cultures con­tain—independent of their source—different types of by-products. Separa­tion of all these substances may be necessary because of the possibility of undesired effects.

Considering enzyme stability there is another reason for treatment of crude enzyme preparations. Since the trend in enzyme applications is to­ward use of liquid preparations, stabilization is an important procedure.

Techniques for the large-scale isolation and (partial) purification of enzymes from micro­bial sources make use mainly of traditional procedures. Most of the equip­ment can be found in food-processing plants. Large-scale equipment specific for enzyme isolation is not marketed.

Nearly all process operations are carried out at low temperatures (prefer­ably 0°-10°C), with the exception of drying.

Separation processes are usually conducted in batches rather than con­tinuously. However, the scale-up of batch operations inherently causes extended processing times which for many enzymes result in increased losses of activity due to denaturation of the enzyme protein.

For this reason the application of continuous operations seems to be useful, but the neces­sity for highly reliable machines and ingenious process control delays introduction of continuous methods. In addition the value of continuous processing is lost when a single process step is conducted batch wise, per­haps during precipitation.

Extraction Methods :

The first step in the isolation of enzymes is their extraction. Techniques that fall into this group are employed either to separate enzymes from solid substrate culture or to release enzymes from the interior of microbial cells.

Extraction of Solid Substrate Cultures:

Enzymes produced by solid sub­strate cultivation used to be of the extracellular type. It is therefore easily conceived that extraction of mold brans is rather a washing out process. Countercurrent techniques of percolation are the most frequently used unit operation.

In many cases the mold bran is dried prior to extraction. This is conve­nient when the utilization of the particular enzyme preparation is seasonal. The cultures can be produced in relatively small equipment all the year round, while the extraction is conducted in times of enzyme demand.

On the other hand, it is easily seen that extraction from dried bran will yield solutions with higher enzyme concentrations. And last, drying avoids inter­ference caused by the activity of living cells of fresh cultures. This argu­ment, however may not apply in continuously operated culture plants.

In all cases the extractant is water which, however, may contain acids (inorganic or organic), salts, buffer, or other substances to facilitate solu­bilization of the enzyme or to improve its stability in solution, or to exclude or minimize undesired effects caused by contaminating by-products or microorganisms.

The decision on whether to employ whole cells for a biochemical process or to use isolated enzymes depends on many factors. Technical difficulties and the related cost of large-scale isolation play an important role.

There are a number of methods for cell disruption, as reviewed by Hughes et al. (1971). Chemical and biochemical methods, such as autolysis, treat­ment with solvents, detergents, or lytic enzymes, have the disadvantage of being in principle batch operations. Their conduct is difficult to standardize and optimize. More recommendable are mechanical techniques.

At present, the APV-Manton-Gaulin homogenizer seems to be the most versatile type for cell disintegration. In this machine the cell suspension passes a homogenizing valve at the selected operating pressure and im­pinges on an impact ring. The strong shearing forces combined with the sudden decompression lead to a disruption of the cell wall. Dunnill and Lilly (1972), who examined the disruption of yeast, found that release of protein can be described by a first order rate equation-

Where R is the amount of soluble protein released in g per kg cell mass, Rm the maximum amount of soluble protein released, K a temperature-dependent rate constant, N the number of times the cell suspension has passed the homogenizer, and P the operating pressure. With industrial models, be­tween 50 and 9000 liters of bacterial suspension per hr can be treated, depending on the size of the machine. Ball mills available on the market have a volume capacity of 0.6-250 liters.

Separation Methods :

It is possible here to give only the barest outline of methods that find wider application in the large-scale production of enzymes.

Solids Separation Techniques:

Such methods are involved in the clari­fication of culture liquors and extracts, in the separation of precipitates, and in the sterilization of liquid enzyme preparation by mechanical methods.

The solids to be separated may have a number of properties which make separation processes difficult. For instance, they may be greasy, sometimes colloidal, and often density differences between solid particles and liquid phase are very small. Therefore, pretreatment of the liquor is usually inevitable, as conducted by acidification, addition of water miscible sol­vents or liquid polyions, mild heating, etc.

The problems of large-scale solid-liquid separation are complex and di­verse. There are two approaches- centrifugation and filtration. Industrial centrifuges are not ideal for removal of finely divided biological solids. Disc type centrifuges without solid discharge have proved most efficient for separation of easily settling suspensions of greasy particles. Decanters are used in cases where solids content is high but easily settling, e.g., in the production of dried acetone precipitates.

In cases of poorly settling protein precipitates, hollow bowl centrifuges are employed for separation from low solids suspensions as obtained during fractionated enzyme precipitation. In all cases flow rates must be determined empirically. Sometimes (e.g., with precipitates) the throughput is reduced to less that 10% of the nominal capacity. This requires the integration of cooling devices.

Most frequently filters are more suitable for separation of biological particles. Generally large proportions of filter aids are required. In continu­ous processes vacuum drum filters are used, with diatomaceous earth, wood-meal, or starch as pre-coat materials. Batch operations are conducted with filter presses.

Membrane Separation Techniques:

Membrane processes allow separa­tion of solutes from one another or from a solvent, with no phase change or interphase mass transfer. There are many different kinds of membrane processes, the classification of which is based on the driving forces that cause the transfer of solutes through the membrane. Such a force may be trans-membrane differences in concentrations, as in dialysis electric poten­tial, as in electro-dialysis or hydrostatic pressure, as in microfiltration, ultrafiltration, and reverse osmosis.

At present, ultrafiltration is the only membrane process of importance in large-scale enzyme production. From normal filtration processes it differs just by the size range of the particles to be separated (molecular weight cutoffs between 500 and 300,000). Two types of ultrafiltration membranes are used, which differ in their transport mechanisms and their separation properties.

Isotropic porous membranes are the type most similar to conventional filters. They possess a spongy structure with extremely small random pores the average size of which is in the range of 0.05 to 0.5 microns. Molecules with a diameter smaller than that of the smallest pore will pass the mem­brane quantitatively, whereas particles larger than the largest pore will be retained at the filter surface. Molecules of intermediate size, however, will only pass to some extent.

Another proportion of these particles will be retained within the structure of the membrane. This leads, first, to a decrease in retention (or vice versa, in permeation) with a resulting fouling of the membrane, and secondly, to a reduced discrimination among solutes of different size. In order to minimize fouling it is useful to use membranes with a mean pore size well below that of the solute to be retained. Therefore, porous membranes are advisable for the concentration of high molecular weight solutes (molec. Weight < 1 x 10 6 ).

Diffusive membranes are capable of more selective molecular discrimina­tion. They are essentially homogeneous hydrogel layers, through which the solvent as well as the solute is transported by molecular diffusion under the driving force of a concentration or a chemical potential gradient. The transportation of a molecule through the membrane requires considerable kinetic energy.

This depends, of course, on the dimensions of the diffusing molecule and on the mobility of the single polymer chains within the membrane matrix. As a rule, the rate of diffusion is high when the polymer segments of the matrix are only loosely interlaced, i.e., when the gel matrix is highly hydrated. For this reason all membranes made from hydrophilic polymers and capable of swelling in water to a certain degree are princi­pally suited as pressure filtration membranes for aqueous solutions.

In addition to the retention potential of the membrane the flux is impor­tant for economic reasons. Since in both the porous and the diffusive filter types the flux depends largely on the thickness of the membrane, it is necessary to keep the membrane as thin as possible.

This requirement has been fulfilled by the construction of anisotropic membranes. They consist of a highly consolidated but very thin (0.1-5 μm) active layer on a compara­tively thick (1 to 20 microns) highly porous support. The advantage of these anisotropic membranes is that there is no reduction in solvent permeability at constant hydrostatic pressure because there is no blockage within the membrane.

In any ultrafiltration system, accumulation of solutes at the membrane surface occurs, which leads to formation of a “slime” that increasingly impedes solvent flow through the membrane, until convective transport of solute toward the membrane is equal to the rate of back diffusive transport away from the membrane. This phenomenon is called “concentration polar­ization”.

Proteins and colloidal particles build up solid or thixotropic gels when concentrated beyond a certain point. The solute concentration on the membrane surface reaches an upper limit which is typically between 20 and 70% solute by volume. In order to reduce the polarization effect, in industrial ultrafiltration equipment the feed solution passes the membrane surface at high flow rate.

When a macromolecular solution is ultra-filtered, flux of solvent is de­scribed by the relationship-

Where A is the membrane constant (dependent on temperature, indepen­dent of pressure over the normal operating range), ΔP is the hydrostatic pressure driving force, and Δπ is the osmotic pressure difference across the membrane. For macromolecular solutions with concentrations over 1% w/v, osmotic pressures exceeding 10 to 50 psi are not uncommon.

There are several basic types of ultra-filters – thin channel, tubular, helical tubes, spiral wrapped, and hollow fiber systems. Suitability of a single type depends on the properties of the system to be treated.

The technique of ultrafiltration has the advantage of combining both separation of impurities and concentration of the desired enzyme. However, due to its principle, it is a rather nonselective process. Better results, regarding separation of molecules from each other, can be obtained by gel filtration, but in many cases its application is not economically feasible.

In principle, gel filtration is the diffusional partitioning of solute mole­cules between the readily mobile solvent phase and that confined in spaces within the porous gel particles that make up the stationary phase. Diffusional exchange of solutes takes place between the stationary and mobile phases. The extent to which a molecule penetrates the station­ary phase is represented by the partition coefficient Kav, according to the equation

Where Ve is the volume of solvent required to elute solute from the gel column, V0 is the void volume (i.e., the volume of liquid external to the gel particles), and Vt is the total volume of column bed. Kav is inversely proportional to the log of the molecular weight, as shown empirically.

Gel filtration works rapidly and preservingly, without mechanical stress as in ultrafiltration. However, precipitation of proteins within the gel column may occur as a consequence of desalting. In some cases it has been observed that the metal bridges of enzyme quaternary structures were uncoupled.

Adsorption Techniques:

Because of the possibility of highly selective separations, adsorption processes are increasingly used. Properties of en­zyme molecules as different as, e.g., lipophily, electric charge, specificity, etc., are the basis of separation. This results in a great number of adsor­bents, such as active carbon, hydroxyapatite, ion exchangers, carrier fixed substrate analogs, and so forth.

A common feature of all adsorption tech­niques is the principle of adsorption followed by desorption or elution. Separation is achieved by adsorption and elution of either enzyme or impu­rities. Two methods are available, batch wise adsorption or column chroma­tography. The latter process has greater separation efficiency and, in addi­tion, offers the possibility of semi-continuous operation.

Among the different adsorption techniques, affinity chromatography is of very great interest, but far from being applicable on a large scale. Ion exchangers available for large-scale processes are of the resin, large-pore gels, or cellulose types. In particular, ion exchange resins exhibit useful properties for industrial production of enzymes.

It must, however, be taken into consideration that the proximity of a resin matrix with a high charge density can affect the structural integrity of enzymes. Large-pore ion exchangers and cellulose exchangers have a number of properties which make them very suitable for enzyme separation processes, but the former are very costly. Obviously, they are only suitable for batch processes be­cause they are compressed in columns.

Precipitation Techniques:

Separation from solution by salting out is one of the oldest and yet most important procedures of concentration and purification of enzymes. The logarithm of the decrease in protein solubility in concentrated electrolyte solutions is a linear function of increasing salt concentration (ionic strength), as described by the equation-

where s is the solubility of the protein in g/liter solution τ the ionic strength in moles per liter B 1 , an intercept constant, is dependent on pH, tempera­ture, and the nature of the protein in solution K 1 is the salting out constant which is independent of pH and temperature, but varies with the protein in solution and the salt used. From the preceding relationship it can be derived that precipitation of protein of known concen­trations will occur when the ionic strength satisfies the equation

This means that the electrolyte concentration required for protein precipi­tation varies with protein concentration.

The influence of the most important precipitation parameters can be outlined shortly as follows – Higher valency salts produce higher ionic strength than lower valency salts. At a constant ion strength, protein solubility increases with increasing distance (in both directions) from its isoelectric point. As a result, lower ionic strength is required for precipita­tion when carried out at the isoelectric point of the protein.

The commonly used salt for precipitation is ammonium sulfate. The reasons can be found in the high solubility of this salt and in its low price. In addition, ammonium sulfate is nontoxic for most enzymes and in many cases it acts as a stabilizing agent. In ammonium sulfate solutions precipitated enzymes are often storable for years without significant loss when kept at low temperatures.

In contrast to neutral salts, solvents are less customary for large-scale precipitation of enzymes. The reason is higher costs of raw materials and equipment. Explosion proof equipment and recycling of the solvents are inevitable requirements.

Solvent precipitation is based on the fact that the solubility of enzymes decreases with the decreasing dielectric constant (ϵ) of the solvent. The concentration required is lower the less hydrophilic the solvent is. Thus, an increasing precipitating effect can be achieved in the series methanol (ϵ25 = 33), ethanol (ϵ25 = 24), isopropanol (ϵ25 = 18). Besides aliphatic alcohols, acetone (ϵ25 = 20) is often used as a precipitant.

Solvent precipitates are distinguished from salt precipitates by the ease with which they settle. However, at temperatures above 4°C denaturation of the enzyme protein can occur. Therefore, it is quite normal to work at temperatures below zero. This, however, requires large cooling capacity in industrial manufacture.

All these difficulties can be avoided by using polyethyleneglycol with a molecular weight of 6000. This precipitant does not effect enzyme denatur­ation and is relatively independent of temperature and electrolyte concen­tration. However, there is a strong dependence on hydrogen ion concentra­tion. The best results are obtained at the isoelectric point of the enzyme to be precipitated.

As the solubility of a protein molecule is lowest at its isoelectric point, successive precipitation of different enzymes from a solution can be achieved by changing the pH. These precipitates settle easily and can easily be separated from the solution by centrifugation.

Conversion to Storage Form :

Storability of an enzyme requires the preparation of a suitable storage form. Commercial enzyme products are available either in solution or in solid state. Generally, users prefer solutions because of their easier han­dling, but enzymes are usually very unstable in aqueous solution.

For this reason stabilization of dissolved enzymes is a very important step in the manufacture of liquid enzyme preparations. The storage stability is af­fected by the following two factors- microbial deterioration of the enzyme solution and denaturation of the enzyme protein. These two problems seem to be closely related to each other.

Many treatments have been tried in order to prevent growth of microor­ganisms. The methods include, for instance, incorporation of chemical pre­servatives, pasteurization, addition of salts and polyhydric alcohols, and irradiation. But some of those treatments are undesirable due to legal aspects. Therefore, the most suitable method to repress microbial growth is to dissolve the enzyme in a highly concentrated solution of salts and sugars.

With liquid preparations, storage at low temperatures and at suitable pH is essentially inevitable. It is well known that substrates almost invariably protect the corresponding enzyme against physical, chemical, or physicochemical agents. This can be attributed to either conformational stabiliza­tion or steric or competitive protection.

From a number of publications it can be seen that almost any effect on enzyme stability may a priori be an allosteric one due to attack at sites other than the active site of the enzyme. For example- in thermolysin, a bacterial protease, Ca ions stabilize the enzyme molecule, while Zn ions are required for activity. The enormous value of Ca ions in stabilizing bacterial α-amylase has long been known.

A number of techniques are available for stabilization.

Some of them are presented in the following list, immobilization methods excluded:

(1) Conformational or charge stabilization and/or protection from dilution-dissociation by using buffers, glycerol, substrates, or inhibitors.

(2) Protection of active site thiol via disulfide exchange by thiols, redox dyes, oxygen-binding agents, or chelating agents.

(3) Miscellaneous methods include, e.g., inhibition or removal of proteo­lytic enzymes protection from light by photosensitive dyes lowering activity of water by viscosity effectors, salts, or sugars lowering surface energy by antifoams cooling and crystallization protection by antifreeze removal of harmful agents and sterilization for protection against microbial attack.

Commercially available solid enzyme preparations are dried mold brans, dried precipitates, or dried solutions. Spray drying is the preferred method for removal of water from enzyme solutions due to economic reasons. How­ever, it is only applicable to enzymes sufficiently resistant to the tempera­ture conditions of this process. On the other hand, freeze drying is most preserving, but its use is limited by cost considerations as well as by the fact that unless the salt concentrations of the enzyme solution are sufficiently reduced, eutectic mixtures may be formed.

This may lead to incomplete drying or to severe foaming and protein denaturation. A specific method of drying sometimes used is granulation in a fluidized bed with milk sugar or maltodextrin as carrier. In this case, of course, sufficiently high specific activity of the enzyme is required in order to ensure satisfactory activity of the commercial preparation.

Enzyme Immobilization :

In commercial applications enzymes are used commonly in the soluble or “free” form. This practice, however, is very wasteful, because the enzyme is discharged at the end of the reaction, although its activity is scarcely lessened in reactions carried out under optimum conditions.

Immobiliza­tion prevents diffusion of the enzymes in the reaction mixture and permits their recovery from the product stream by simple solid-liquid separation methods. As a consequence, reaction products are free of enzyme and reuse of the enzyme is possible. Another advantage of immobilized enzymes is that they can be used in continuously operated reactors.

Methods of Immobilization:

In principle, immobilization of an enzyme can be achieved by fixing it on the surface of a water-insoluble material, by trapping it inside a matrix that is permeable to the enzyme’s substrate and products, and by cross-linking it with suitable agents to give insoluble particles. Bound enzymes may be prepared by covalent coupling to active matrices or by heteropolar and/or van der Waals binding to adsorbents or ion ex­changers.

Covalent coupling to activated carrier materials is achieved by methods known in peptide and protein chemistry. Some examples of enzymes immo­bilized in this pattern are given in Table 15.3. The formation of covalent bonds has the advantage of an attachment which is not reversed by pH, ionic strength, or substrate. However, covalent binding offers the possibil­ity that the active site of enzyme may be blocked through the chemical reaction used in the immobilization reaction and the enzyme rendered inactive.

There are a large number of methods of covalent attachment. The groups of enzymes that take part in the formation of the chemical bond are- amino, imino, amide, hydroxyl, carboxy, thiol, methylthiol, guanidyl, imidazole groups, and the phenol ring. Methods have been developed for covalently attaching enzymes to inorganic carriers such as alumina, glass, silica, stainless steel, etc.

Adsorption of enzymes at solid surfaces (Table 15.4) offers the advantage of extreme simplicity. It is carried out accord­ing to the principles of chromatography. The conditions of adsorption in­volve no reactive species and thus do not result in modification of the enzyme. The binding of enzymes, however, is reversible and for this reason adsorbed enzymes present the problem of desorption in the presence of substrate or increased ionic strength.

Commonly used adsorbents include many organic and inorganic materials such as alumina, carbon, cellulose, clays, glass (including controlled-pore glass), hydroxyapatite, metal oxides, and various siliceous materials. Ion exchange resins bind enzyme by electrostatic interactions. The first successful commercial application of immo­bilized aminoacylase (for resolution of DL-amino acids) involved fixing of the enzyme by adsorption to DEAE-Sephadex as carrier.

Inclusion of enzymes in polymer gels, microcapsules, or filamentous structures has the advantage of relatively mild reaction conditions. This method is free from the risk of blocking active site groups on the enzyme molecule by chemical bonds the enzyme is retained in its native state.

The major drawbacks of this immobilization technique are two- retardation of the enzymic reaction due to diffusional control of the transport of substrate and products (particularly with high molecular weight substrates and/or products) and continuous loss of enzyme due to the distribution of pore sizes. Materials used for entrapment include silicone rubber, silica gel, starch, and, preferably, polyacrylamides. Exam­ples of enzymes immobilized by entrapment are given in Table 15.5.

A variation of the inclusion method is encapsulation within semiperme­able membranes. Materials such as collodion poly­styrene, cellulose derivatives, and, most commonly, nylon have been used to form thin, spherical, semipermeable membranes shaped into micro­capsules which include the enzyme to be immobilized. The size of the capsules can range from μm to many μm. As has been demonstrated by Kitajima and Kondo (1971) with yeast, it is possible to encapsulate multi-enzyme systems from cell extracts and to carry out fermentation in such artificial cells.

Enzymes can be polymerized by cross-linking with low molecular weight multifunctional agents (see Table 15.6). This method leads to the formation of a three-dimensional network of enzyme molecules when the reaction is carried out in the absence of a support. However, usually it results in a considerable loss of activity. Com­monly, enzymes are cross-linked after adsorption onto a suitable carrier.

Cross-linking agents most commonly used include diazobenzidine and its derivatives and particularly glutaraldehyde. On the other hand, enzymes can become immobilized by copolymerization, i.e., covalent incorporation into polymers. The methods most often em­ployed involve copolymerization with maleic anhydride and ethylene. As with entrapped and microencapsulated enzymes, these derivatives show little or no activity toward macromolecular substrates.

An alternative to the immobilization of isolated enzymes is immobiliza­tion of whole microbial cells. This method provides a means of avoiding expensive enzyme purification operations. Entrapment of enzymes within whole cells may also be useful when various enzymes are involved in a given process.

And, finally, immobilized intact cells have proved effective in processes involving enzymes that require cofactors for mediating their catalytic action. Some examples of whole cell immobilization are shown in Table 15.7. Cells can be immobilized by fixing them to carriers, such as fibers or granular materials, or by entrapment.

Properties of Immobilized Enzymes:

After immobilization of an en­zyme, its properties can be changed significantly. Such alterations may be attributed to (1) the physical and chemical nature of the carrier used, (2) the chemical and/or conformational changes in the enzyme structure, and (3) the “heterogeneous nature” of catalysis caused by immobilization.

Effects of altered reactivity include kinetic constants (resulting from a change in activation energy), optimum pH, Michaelis constant, and sub­strate specificity. A matrix charge can affect the hydrogen ion concentra­tion in the locus of the attached enzyme and thus change its apparent pH optimum in one direction or the other, depending on the use of either cationic or anionic carriers, and the apparent Michaelis constant if the substrates are also charged it is increased if the matrix and the substrate charges are alike and decreased if they are opposite. Changes in enzyme specificity can result from conformational changes in the enzyme molecule caused by the attachment itself.

One of the more important results of enzyme immobilization is the reten­tion of activity for considerable periods of time under suitable conditions of storage. The stability of immobilized enzymes to storage, heat, and pH basically depends on the nature of the carrier surface to which the enzyme is bound.

Among 50 immobilized enzymes, as compared with their soluble counterparts, Melrose (1971) found 30 more stable and 8 less stable than the soluble forms 12 showed no difference from the free systems. The reasons for the observed increase in stability are not clear. It may be attributed, for example, to prevention of conformational inactivation or to shielding of active groups on the enzyme from reactive groups in solution.

How Enzymes Work

Figure 3. Diagram of a catalytic reaction showing difference in activation energy in uncatalysed and catalysed reaction. The enzyme reduces the energy barrier required to activate the substrate, allowing more substrates to become activated, which increases the rate of product formation. Note that the energy difference between the substrate and the product is not changed by the enzyme.

In all chemical reactions, there is an initial input of energy that is required before the reaction can occur. If this initial energy requirement (called the activation energy or energy barrier) is small, then the reaction will happen quickly and easily. If the activation energy is large, then the reaction will take longer to occur. Enzymes function to reduce the activation energy required for a chemical reaction to occur.

First, the enzyme binds to the substrate and slightly distorts its shape. The change in shape activates the substrate molecule and decreases the total activation energy required for the substrate to be turned into product. As the number of activated substrate molecules increases, so does the conversion of substrate to product. An analogy for this effect is a ski hill, with skiers at the bottom of one side of the hill representing substrates, skiers on the top of the hill representing activated substrates, and the products being the number of skiers that ski down the other side. If the height of the hill is lowered (due to the presence of the enzyme), then more skiers can make it to the top, increasing the number that ski down to become products.

Practice Questions

Fill in the blank: When an enzyme catalyzes a reaction, ________.

  1. it raises the activation energy of the reaction.
  2. it is used once and discarded.
  3. it becomes a product.
  4. it acts as a reactant.
  5. it lowers the activation energy of the reaction.

What will happen to the rate at which a chemical reaction proceeds if the activation energy is increased?

  1. The reaction will happen faster (at a higher rate).
  2. The reaction will happen slower (at a lower rate).
  3. The reaction rate will not change.

In Summary: Enzymes

Enzymes are proteins that speed up reactions by reducing the activation energy. Each enzyme typically binds only one substrate. Enzymes are not consumed during a reaction instead they are available to bind new substrates and catalyze the same reaction repeatedly.

Key Pathway • Ras/RAF/Mitogen-Activated Protein [MAP] Kinase Pathway

Ras = G-protein specific to this pathway RAF = proto-oncogene serine/threonine kinase

MEK = mitogen-activated protein kinase MAPK = mitogen-activated protein kinase

Ras • a small G protein (GTPase) involved in signal transduction leading to cell division and proliferation. If not regulated properly, Ras proteins can lead to uncontrolled cell division that eventually results in tumor formation.

RAF [Rapidly Accelerated Fibrosarcoma] • family of protein kinases that are involved with retroviral oncogenes (genes that can potentially cause cancer).

Tyrosine-Kinase Associated Receptors • associate with intracellular proteins that have tyrosine kinase activity. These receptors lack the tyrosine kinase domain that was discussed earlier and, therefore, accomplish tyrosine phosphorylation by cytoplasmic tyrosine kinases instead.

Cytokine receptors make up the largest family of receptors that relay signals into the cell by cytoplasmic tyrosine kinases. These particular receptors are associated with the cytoplasmic kinase, Jak (Janus kinase). Jak will go on to activate a gene regulatory protein called STAT (signal transducers and activators of transcription). The pathway is described and depicted below:

Key PathwayJak/Stat pathway (Janus kinase/signal transducers and activators of transcription) • The Jak/Stat pathway is the principal pathway for cytokines and growth factors in humans. This pathway is activated by a number of cytokines (most commonly interferons) and growth factors. Activation stimulates cell proliferation, differentiation, migration and apoptosis. Furthermore, cytokines control the synthesis and release of a number of inflammatory mediators. When a cytokine binds to its enzyme-linked receptor it results in a conformational change leading to phosphorylation of the intracellular active-enzyme domain, eventually leading to the transcription of inflammatory mediators. As with all signal transduction mechanisms, homeostasis is reliant on proper regulation of all these different pathways. Lack of proper regulation of the JAK pathway can cause inflammatory disease, erythrocytosis, gigantism and leukemias.

ER/SR Calcium Pump: Function

Ca 2+ Binding and Catalytic Activation

Binding of two calcium ions per ATPase molecule is an absolute requirement for enzyme activation . The cooperative character of binding is consistent with a sequential binding mechanism to two interdependent sites (I and II). Spectroscopic studies provided early suggestions of Ca 2+ -induced conformational effects, accounting for binding cooperativity and enzyme activation. A detailed characterization of the large conformational changes produced by Ca 2+ binding was then obtained by high-resolution diffraction studies.

The functional role of the Ca 2+ -induced conformation change lies in its absolute requirement for enzyme activation, rendered possible by the long-range intramolecular linkage. The requirement for Ca 2+ involves both ATP use for phosphoenzyme formation in the forward direction of the cycle and the formation of ATP upon addition of ADP to phosphoenzyme formed with Pi in the reverse direction of the cycle. Activation is not obtained by Ca 2+ occupancy of the first binding site only but requires the occupancy of the second site, which may then be considered a Ca 2+ trigger point for enzyme activation. Ca 2+ -binding affects directly the M4, M5, and M6 transmembrane helices and is then transmitted to the extramembranous region, resulting in the large displacement of the headpiece domains and catalytic activation. Single mutations of several residues in the segments connecting the Ca 2+ -binding region with the phosphorylation domain, such as M4, M5, and the M6–M7 loop, interfere with the phosphorylation reactions.

C3 and C4 Plants

Carbon fixation resulting in a three-carbon sugar is known as C3 photosynthesis, and most plants use this kind of photosynthesis. It works well in cool, moist conditions where plants can keep the pores that let carbon dioxide in, called stomata, open throughout the day.

For many summer annual plants, conditions are too hot to allow stomata to remain open throughout the day, so these plants have adapted by developing C4 photosynthesis. C4 photosynthesis is named as such because the fixation of carbon dioxide, by an enzyme called PEP carboxylase, results in a four-carbon sugar. The carbon acquired this way is then passed on to RUBISCO and enters the Calvin cycle.